Congenital Heart Disease (CHD) is the most common developmental anomaly, occurring in 1-3% of live human births. CHD often manifests as a primary structural defect, however, palliation of these malformations is frequently incomplete, and can result in the development of cardiomyopathy and heart failure later in life. Atrioventricular septal defects (AVSDs) are a common and particularly severe form of structural CHD, characterized by a common atrioventricular junction, and are often caused by malformation of the atrial septum (AS). The AS is derived from a population of cells called the posterior Second Heart Field (pSHF), a heterogeneous subset of progenitors that contributes to the developing inflow tract after the initial formation of the cardiac chambers. Our lab and others have demonstrated a requirement for functional transcription factors (TFs) and active signaling components, including those of the Hedgehog (Hh) signaling pathway, for normal atrial septum formation from the pSHF in mice. Preliminary gene expression analysis performed in our lab has demonstrated that a lack of Hh signaling in the pSHF of E10.5 embryos leads to a downregulation of several progenitor-specific genes, and an upregulation of genes characteristic of cardiomyocyte differentiation. We have also now demonstrated that the positive Hh TF, Gli1, is absent from differentiating cardiomyocytes and the repressive Hh TF, Gli3T, is present in differentiating cardiomyocytes in vitro. Finally, we show that chromatin profiling can be used to identify a network of progenitor-specific, Hh TF-controlled distal regulatory elements. We hypothesize that Hh signaling may function to regulate Foxf1a and additional progenitor genes in the pSHF via distal regulatory elements through a switch mechanism whereby the presence of active Hh signaling leads to the upregulation of genes required for the maintenance of a progenitor state, and a lack of Hh signaling represses these genes, leading to cardiomyocyte differentiation. This proposal will define the mechanisms by which Hh signaling in the embryonic second heart field directs the formation of the atrial septum, and illuminate the network through which Hh TFs control the timing of cardiac progenitor differentiation. Defining these progenitor transcriptional networks will allow for better prediction of AVSD risk and inform a strategy for primary prevention.